The long-term goals of this project are to identify the specific subcellular processes that intervene between luteinizing hormone releasing hormone (LHRH) - receptor interaction and the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary gland and to determine how these processes are regulated by LHRH and modulated by other hormones. In the proposed studies, we will examine calcium movement into the gonadotrope and the consequence to the immediate gonadotropin secretory event and to the self priming response. In addition, the secretory granule will be examined as one site at which regulation of both immediate release and self priming may converge. The specific aims are to: 1) Establish a pituitary cell culture system for assessing rapid changes in LH and FSH secretory activity as well as LHRH self priming and for performing in situ radiolabeling studies. Superfusion of pituitary pieces and of pituitary cells cultured on coverslips including cultures enriched for gonadotropes will be used. 2) Identify the specific calcium channel types modulated by LHRH which are involved in the secretory event in the gonadotrope. The probes for this will be a series of divalent cations of varying channel permeabilities and channel-specific blockers; the endpoints will be changes in gonadotropin secretory activity. 3) Determine the consequence of a change in cytosolic (calcium) to selected intracellular mediators of LHRH action. Cyclic AMP generation will be used as a measure to compare cAMP-mediated regulation of LHRH self priming with the augmentation of secretion stimulated by barium and zero calcium/EGTA media. The ability of barium and other divalent cations to affect the activity of protein kinase C and one of its substrates will be evaluated. 4) Determine the characteristics and regulation of p36 and p35 (protein kinase C and tyrosine-protein kinase substrates in pituitary secretory granules). This will include establishing the identity of pituitary p36/p35 with calpactinI/calpactinII by immunoblotting, tryptic peptide mapping and phosphoamino analysis, defining the calcium/phospholipid dependent association of p36/p35 with secretory granules and plasma membranes in the gonadotrope, and identifying the agonist-stimulated pathway regulating phosphorylation of p36/p35 in the pituitary gland. These studies will provide insight into the mechanisms of action of LHRH in that modulation of specific intracellular events will be related to physiological effects of the hormone.